Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a polypeptide found in many mammals. GM-CSF is a lymphokine that stimulates the proliferation of a variety of undifferentiated progenitor cells involved in the immunological response. Various cellular components of the bone marrow are known to be stimulated by GM-CSF. See, MacDonald et al., J. Bone Mineral Res., 1 (2):227 (1986); Begley et al., Exp. Hematol., 13:956 (1985). Complementary DNAs (cDNAs) for GM-CSF have recently been cloned and sequenced by a number of laboratories, e.g., Gough et al., Nature 309:763 (1984), PCT Application No. 85/04188 (mouse); Cantrell et al., Proc. Natl., Acad. Sci 82:6250 (1985); European Patent Application No. 183,350 (human). Moreover, non-recombinant GM-CSF has been purified from various culture supernatants, e.g., U.S. Pat. No. 4,438,032 (Mo cell line); Burgess et al., Exp. Hematol. 9:893 (1981) (mouse); Sparrow et al., Proc. Natl. Acad. Sci., 82:292 (1985) (purification and partial amino acid sequence for mouse); Wu et al., Exp. Hematol., 12:267 (1984) (rat); Gasson et al., Science 230:1171 (1985) (human); Burgess et al., Blood, 69:43 (1987) (human). Among the human GM-CSFs, nucleotide sequence and amino acid sequence (primary structure) heterogeneity have been observed. For example, at the amino acid level of human GM-CSF, both threonine and isoleucine have been observed at position 100 with respect to the N-terminal alanine, suggesting that allelic forms, or polymorphs, of GM-CSF may exist within human populations.
Also, various leader sequences occur at the N-terminal portion of the amino acid sequence. These leaders may be of various lengths and amino acid composition, which may or may not affect biological activity. Often the leader sequence has a methionine residue at its N-terminal end. The native mature protein for GM-CSF is about 127 amino acid residues long. See PCT Application No. 86/03225 and PCT Application No. 86/00639 for comparisons of human GM-CSF sequence to the mouse and gibbon sequences, respectively.
Details about the secondary and tertiary structure for GM-CSF have yet to be elucidated, although a hydrophilicity plot for human GM-CSF has been constructed by Hopp and Woods [Proc. Natl. Acad. Sci. USA, 78:3824-8(1981)]. Many details about the mechanism of GM-CSF-induced cell proliferation remain a mystery.
Nevertheless, GM-CSF has been implicated as a factor in a number of disease states. The presence of elevated levels of GM-CSF concomitant with various disease states suggests that GM-CSF may have a role in autocrine or paracrine control of the growth of aberrant cells. Such control has been observed and/or postulated for leukemia/lymphomas, solid tumors, metastatic lesions, diseases involving macrophage infiltration, and cyclic neutropenia. It has been observed that the transformation of non-leukemogenic hematopoietic cell lines to the malignant phenotype was associated with the capacity to synthesize colony-stimulating factors (Hapel, et al. 1981; Schrader and Crapper, 1983). The hypothesis that autocrine stimulation by GM-CSF can result in leukemogenicity has been directly tested by using a retroviral vector to express GM-CSF in FDC-P.sub.1, a factor-dependent murine cell line (Lang, R. A., Metcalf, D., Gough, N. M. et al., 1985), (see below). In these cases the colony-stimulating factor was IL-3 (multi-CSF). Thus, their results do suggest that appropriate antagonists for the CSF's could be employed as anti-leukemogenic agents.
The observation of constitutive expression of GM-CSF in patients with acute myelogenous leukemia (AML) [Young et al., J. Clin. Invest., 79:100-106 (1987)] has lead several investigators to urge caution [Begley et al., Leukemia, 1:1-8 (1987)] in the therapeutic application of GM-CSF in the treatment of AML and perhaps other diseased states.
Autostimulatory and autocrine synthesis have been implicated in the mechanism of oncogenesis of myeloid cells. Autologous production of particular cytokines resulting in autostimulation is believed to be a critical oncogenic step (Schrader et al., J. Cell Biochem. Abstract, 1988). Schrader et al. do suggest, however, that the "aberrant activation of lymphokine genes may be a common mechanism of oncogenic progression" given the appropriate target cell. It is consistent with this proposal that in a study of 24 cases involving myeloid leukemic cells, Mannoni et al., J. Cell Biochem. Abstract, (1988) report that these cells respond to GM-CSF by proliferating but not by differentiating.
Experimental induction of autocrine synthesis of GM-CSF [Gonda et al., Cell 51:675-686 (1987)] suggest that autocrine growth may be an important step in the progression to fully developed leukemia.
Studies demonstrating a direct relationship between autocrine stimulation by GM-CSF and leukemogenicity were based on the use of a retroviral vector to express GM-CSF in FDC-P.sub.1, a factor-dependent murine cell line (Lang, R. A., Metcalf, D., Gough, N. M. et al., Cell 43:531-542, 1985). Virally infected cells were shown to synthesize GM-CSF and to proliferate in the absence of exogenous GM-CSF. This result contrasts with data obtained for the uninfected cells, which require exogenous IL-3 or exogenous GM-CSF for survival and proliferation. In addition, it was observed that the infected cell lines caused large, diffusely infiltrating tumor masses in syngeneic DBA mice. In distinction, no animals injected with FD cells developed transplanted leukemias during an observation period of 26 weeks. Thus, simply changing the ability of FD cells to synthesize GM-CSF appears to be sufficient to convert them to a tumorigenic phenotype.
Studies have been carried out by Laker et al., Proc. Natl. Acad. Sci. USA, 84:8458-8462 (1987) in which transfer of the GM-CSF gene into factor-independent cell lines resulted in the acquisition of factor-independent growth with an intermediate requirement for external stimulation followed by a second mutation which eliminated the need for external GM-CSF. The rate of transition of growth independence was highly correlated with the levels of GM-CSF produced, but could not be shown to be dependent upon a threshold level of GM-CSF. These studies suggest that the two steps of autocrine synthesis and autonomous growth were distinct but were interconnected in the progression toward a malignant state.
Antisera to GM-CSF significantly inhibit "spontaneous" growth associated with juvenile chronic myelogenous leukemia [Gualtieri et al., Clin. Res. 36(1):24A (1988)]. Santoli et al., J. Immunol. 139: 3348-3354 (1987) and Valtiei et al., J. Immunol. 138: 4042 (1987)] have established a T-lymphocytic leukemia derived cell line Tall 101. GM-CSF was shown to support the long-term growth of this cell line and to act synergistically with IL-3 to stimulate proliferation of T-lymphoblastic leukemia.
Constitutive expression of GM-CSF has been detected in several solid tumors. Evidence for the involvement of GM-CSF in tumor development is as follows.
Expression of GM-CSF in a lung squamous cell carcinoma was observed by Mano et al., Japan. J. Cancer Res., 78:1041-1043 (1987).
Binding of GM-CSF to a single class of high affinity receptors on small cell carcinoma cell lines has been reported by Baldwin et al., J. Cell Biochem. Abstracts (Suppl. 12A):97, (1988). In this same study these cell lines were shown to respond to GM-CSF in an in vitro colony growth assay.
Constitutive expression of GM-CSF was observed by Mano et al., Japan J. Cancer Res. 78:1041-1043 (1987) in a cell line established from lung large cell carcinoma derived from a patient. Constitutive expression of GM-CSF was observed by Mano et al., Japan J. Cancer Res. 78:1041-1043, (1987) in a cell line derived from a 43 year old male patient with a white blood cell count of 9000-10000/mm.sup.3.
Dedhar and Gallaway, Abstracts of the Seventy-Ninth Annual Meeting of the American Association for Cancer Research, p. 51 (1988) and Dedhar et al., J. Cell Biochem. Abstracts (Supply. 12A):128 (1988) have reported that GM-CSF promotes proliferation of human osteogenic sarcoma cell lines MG-63 and HOS.
In studies by Hayashi et al., J. Cancer Res. 78: 1224-1228 (1987), a human bladder carcinoma cell line (HTB9) was found to express mRNA of the GM-CSF gene.
Ensoli et al., Congress on Cytokine Res. (1988) have reported that Kaposi's sarcoma (KS) cell lines derived from AIDS-KS patients express "abundant" levels of mRNA for GM-CSF. In describing a potential mechanism for autocrine growth stimulatory role in the initiation of KS-lesions, Biberfeld et al., J. Cell Biochem. Abstracts, p. 143 (1988) have noted the significant levels of several growth factors including that of GM-CSF.
Available data suggest that the metastatic process, particularly at late stages, may be enhanced by GM-CSF production. For example, an enhancement of lung metastases has been observed in mice which had a marked granulocytosis [Ishikawa and Ziff, Arthritis and Rheumatism 19: 1-3 (1976)]. Similarly, Glaves, Invasion Metastasis 3: 160 (1983), has reported data indicating that PMNs cause an accelerated pulmonary clearance of arrested melanoma cells, In vitro production of GM-CSF by a metastasizing cell line (TS/A) derived from a spontaneous mouse mammary carcinomas has been described [Nicoletti et al., Bio. J. Cancer, 52:215 (1985)], and a relationship was established in these studies between in vitro production of colony-stimulating factor and the capacity for spontaneous metastases. In a further recent series of experiments Nicoletti et al., Anti-cancer Res. 7: 695-700 (1987) investigated colony-stimulating factor production in various TS/A cell variants isolated by serial in vivo selection of lung metastatic nodules.
Studies in which transgenic mice carrying the murine GM-CSF gene expressed from a retroviral promoter resulted in elevated levels of GM-CSF in serum, urine, peritoneal cavity, and eye [Lang et al., Cell, 51:675-686 (1987)]. The mice developed lesions containing macrophages in striated muscle, and accumulations of macrophages in the eyes and in the peritoneal and pleural cavities. A high death rate was attributed to muscle wasting due to macrophage activation.
The following is a list of diseases related to macrophage infiltration in which a GM-CSF antagonist may be useful.
Firestein and Zvailfler, Arthritis and Rheumatism, 30, 857-863 (1987) have sought the factor responsible for monocyte activation in the phenotypes of peripheral blood monocytes and synovial fluid monocytes of patients with chronic inflammatory arthritis. The observation that there were only low levels of gamma interferon in synovial fluid and synovial tissue suggests that gamma interferon was likely not responsible for this activation [Firestein and Zvaifler, Arthritis and Rheumatism, 30, 864-871 (1987)].
The increase in the number of macrophages in the skin is important in cutaneous disorders. Diseases which fall into this category include infectious diseases such as Leishmaniasis and leprosy, and non-infectious diseases such as sarcoidosis and granuloma and annulare. Studies undertaken by Chodakewitz et al., J. Immunol. 140:832-836 (1988), have shown constitutive expression of GM-CSF by keratinocytes, which may play a role in the regulation of cutaneous macrophage responses. Danner et al., J. Invest. Derm., 89:339-340 (1987), have postulated, based on studies of neutrophil activation and oxygen radical release, that GM-CSF may play a role in the inflammatory skin diseases in which neutrophil activator is a "prominent feature".
Consistent with this possible role is the observation by Griel et al., Abstract of XIXth Meeting of the Society of Immunology, (1988), that the systemic treatment by GM-CSF of mice infected with Leishmania major enhances the parasitic burden rather than eliminating it. Thus, diseases associated with accumulations of macrophage aggregates and mononuclear phagocytes, such as sarcoidosis and the other diseases mentioned above, may be good targets for treatment with antagonists of GM-CSF.
Listeria monocytogenes [Cheers et al., Infection and Immunity, 56: 247-251 (1988)] infection was similarly associated with increased serum levels of GM-CSF.
Studies have suggested [Wright et al., Clinical Res. 36: 436A (1988)] that oscillations of neutrophil and monocyte production in the marrow which result in profound neutropenia at regular intervals are due to abnormal responses to GM-CSF by the myeloid progenitors. Several reports have noted marked but transient neutropenia in patients being treated with GM-CSF [Devereux et al., Lancet 121: 1523-1524 (1987)].